Assay for YKL-40 as a marker for degradation of mammalian connective tissue matrices

ABSTRACT

The invention is a method of identifying the presence of a disease state in a mammal which is associated with degradation of connective tissue in the mammal which contains the protein known as YKL-40. The method is a competitive immunoassay for YKL-40. It can be used, for example, to identify the presence of inflammatory or degenerative joint disease and tumor metastasis (to the extent it can be correlated to serum YKL-40 levels). Serum YKL-40 levels as detected and quantified by the inventive method are also suggestive of the prognosis for the length of survival in breast cancer patients following recurrence and/or metastasis of their cancers.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 09/215,077, filed onDec. 18, 1998 now U.S. Pat. No. 6,794,150, which is a continuation ofU.S. Ser. No. 08/581,527, filed on Apr. 17, 1996 now U.S. Pat. No.5,935,798, which is a 371 of PCT/US94/07754, filed on Jul. 8, 1994,which is a continuation-in-part of U.S. Ser. No. 08/089,989, filed onJul. 9, 1993, now abandoned, which are all incorporated herein byreference in their entirety for all purposes.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

This invention was made in part with Government support under Grant No.AR-27029, awarded by the National Institute of Health. The Governmenthas certain rights in this invention.

FIELD OF THE INVENTION

The invention relates to the identification of a circulating proteinassociated with extracellular fiber matrix metabolism in mammalianconnective tissues. More specifically, it is directed to assays for thedetection and quantitation of molecules and fragments of YKL-40, aprotein associated with connective tissue metabolism in mammals. It alsoinvolves correlating serum levels of YKL-40 in a mammal to the presenceand status of diseases in which matrix metabolism plays a role, such asjoint disorders and the metastasis of certain tumors.

BACKGROUND OF THE INVENTION

The extracellular matrix of mammalian connective tissues (such asarticular cartilage of joints and vascular wall tissue of the vascularand lymphatic system) provides strength to, and (to varying degrees) abarrier to the migration of cells from, the tissue. In certain diseaseprocesses, however, the matrix is degraded by hydrolytic enzymes. As thematrix degrades, the integrity of the tissue is impaired, which mayallow tissue cells, by-products and other residues of the matrixmetabolism to escape into bodily fluids and/or lymphatic or vascularcirculation. Detection of these molecules and cells can, in certaininstances, provide information regarding the biochemical characteristicsof the extracellular matrix, including how it is synthesized and how itis lost. Also, where a particular molecule that is produced and/orsecreted during abnormal matrix metabolism is closely related to adisease process, quantitation of that molecule in the patient's bodyfluids and/or tissues can help clinicians monitor the progress of thedisease.

Human joint cartilage is known to contain several different types ofproteins and proteoglycans, a few of which are present only incartilage. These matrix constituents are released from cartilage tissueas it degrades during the course of certain joint diseases. The quantityof released matrix constituents (including fragments thereof and relatedmacromolecules) present in a particular fluid or tissue may correlatewith the intensity of the disease. Conversely, where the damage to thecartilage is reversible (as in secondary reactive arthritis caused byinfection of the joint tissue), a reduction in levels of previouslymeasured released matrix constituents may correlate with the degree ofremission of the disease.

In practice, however, identification of reliable markers for metabolismof cartilage and other connective tissues and development of assays fortheir detection has proved to be a difficult task. Certain releasedfragments and molecules may be rapidly cleared from circulation by thelymph nodes, liver and phagocytosis (see, e.g., Frazer, et al.,Hyaluronan: Sources, Turnover and Metabolism, Clinical Impact of Boneand Connective Tissue Markers 31-49 (Acad. Press, 1989); Smedsrod,“Catabolism in Liver Sinusoids”, id. at 51-73; and, Heinegard, et al.,Brit. J. Rheumatol., 30 (Suppl. 1): 21-24, 1991). Further, certainmolecules are present in several different connective tissues, thusmaking correlation to metabolism in a particular tissue based oncirculating levels of the molecule uncertain. Even where levels of aparticular molecule can be traced to metabolism in the tissue ofinterest, the molecules may decline to undetectable levels or bebiochemically altered in structure during those stages of a disease whena substantial quantity or connective tissue has been lost.

Not surprisingly, therefore, attempts to develop assays, especiallythose utilizing serum, which correlate levels of certain proteins tojoint disease activity have met with mixed success. Rohde and co-workershave described radio-immunoassays (RIAs) for serum levels ofamino-terminal type III procollagen peptide and its degradation productsin rheumatoid arthritis (RA) patients (Rhode, et al. Eur. J. Clin.Invest, 9:451-459, 1979). This propeptide (P-III-NP) can be detected inseveral body fluids; a subsequent report attempted to correlate serumlevels of P-III-NP to disease activity using the Rhode, et al.radioimmunoassay (H.o slashed.rslev-Petersen, et al., Arth. and Rheum.,5:592-599, 1986). While the concentrations of serum P-III-NP weresignificantly elevated in patients with active RA, these concentrationswere also elevated to a similar degree in patient's with inactive RA,thus making the distinction between the two states based on P-III-NPlevels alone difficult.

Assays of serum levels of other connective tissue metabolites andconstituents in RA patients have been attempted in connection withtreatment protocols to gauge the success of those protocols, again withmixed success. For example, H.o slashed.rslev-Petersen, et al., ibid,measured serum levels of P-III-NP, immunoreactive propyl 4-hydroxylaseprotein (1RPH), 7S domain of collagen type IV (Col IV, 7S) and fragmentPI of laminin (S-Lam), which are associated with metabolism ofextracellular interstitial collagens and basement membranes. Althoughserum levels of P-III-NP, 1RPH and Col IV, 7S were elevated in RApatients (as compared with healthy adults), the levels did not declineto normal even with apparent remission of the disease. Also, levels ofS-Lam remained normal in both active and inactive RA patients. As aresult, the presence and quantity of these proteins in serum does notappear to clearly correlate to the progress or remission of RA.

Similar difficulties have also prevented the identification of reliablemarkers for the progress of other connective tissue diseases.Identification of candidate molecules and fragments which may serve asreliable markers for connective tissue metabolism is, therefore, animportant goal of clinical chemistry research. To this end, theexpression of given proteins by matrix-forming cells has been assessedby immunologic assays for antigen and by hybridization assays for mRNAencoding candidate marker protein. Isolation of proteins from theextracellular matrix is, however, limited to the identification ofsecreted proteins that become abundant constituents of that matrix. As aresult, identification of candidate proteins has been limited.

In 1992, the inventors described a method for identification of allproteins secreted by a matrix-forming cell (Johansen, et al., J. Boneand Min. Res., 7:501-512, 1992). Using this method, a 40 kD protein wasidentified as a secreted protein of human bone cells. The inventorshypothesized that the protein (named YKL-40 after the first three aminoacids at the N-terminus and the molecular weight) could play a role inthe action of Vitamin D in bone. YKL-40 appears to be the same proteinidentified by Rejman, et al., (Biochem. Biophys. Res. Commun.,150:329-334, 1988) as being present in mammary secretions ofnon-lactating cows whose mammary glands were undergoing involution

As described in detail below, it has since been discovered that YKL-40can serve as a reliable marker for joint disease, including diseaseswith disparate pathologies such as rheumatoid arthritis andostecarthritis. Surprisingly, it has also been discovered that serumlevels of YKL-40 are also substantially elevated in patients withmetastasis of breast cancer cells, particularly those patients whosurvive for a relatively short period of time following recurrence andmetastasis of their cancer. Further, the inventors have determined thatsignificantly elevated levels of YKL-40 appear in the sera of personshaving connective tissue degradation in organs such as the liver andprostate.

The methods for detecting and quantifying levels of YKL-40 in biologicalsamples described herein, therefore, provide a means of charting theprogress of not only joint disease, but also cancer cell metastasis.Further, based on the apparent relationship of serum levels of YKL-40 toconnective tissue metabolism, it can be reasonably predicted that themethods described will be of use in the diagnosis and monitoring ofother diseases in which connective tissue metabolism plays a role, suchas osteoporosis.

SUMMARY OF THE INVENTION

Detection and quantitation of a marker for diseases whose activity canbe correlated to loss and/or synthesis of connective tissue matrices canbe of value in diagnosing and monitoring the progress of both thedisease and its amelioration. One such marker is YKL-40, a protein ofabout 40 kD molecular weight which has been found in elevatedconcentrations in the blood and synovial fluid of human patients withjoint disease, as well as in the blood of human patients with breastcancer or disorders of organs such as the liver.

A point of commonality between these conditions is their relationship toconnective tissue loss. Specifically, connective tissue loss in jointdisease results from its degradation by enzymes released in the diseaseprocess. In cancer cell metastasis, it is believed that degradation ofthe connective tissue of vessel walls and, possibly, of body organspermits migration of cells from the primary cancer tissue. Similarly,with the loss of organ tissue in an organ degenerative disease such ascirrhosis, it can be expected that connective tissue within the organmay also be degraded.

An object of the invention, therefore, is to provide a method ofdetecting and quantifying YKL-40 in biological samples using an antibodyspecific for YKL-40 and, where appropriate, a detectably labeled antigen(YKL-40).

Another object of the invention is to provide methods for diagnosis ofdiseases which are correlated to the loss and/or synthesis of connectivetissue as indicated by levels of YKL-40 detected in a biological sample.In this respect, the invention is expected to be of particular use inthe diagnosis of joint disease (such as RA), cancer cell metastasis (asin, for example, breast cancer), diseases related to loss of connectivetissue in bone (such as osteoporosis and osteoarthritis), and diseasesrelated to loss of connective and/or other tissue in organs (such ascirrhosis of the liver).

Another object of the invention is to provide methods for thequantitation of levels of YKL-40 to monitor the progress and/oramelioration of a disease which is associated with connective tissuemetabolism related to the presence of YKL-40. Again, the invention isexpected to be of particular use in tracking the progress and/oramelioration of joint disease (such as RA), of cancer cell metastasis(as in, for example, breast cancer), diseases related to the loss ofconnective tissue in bone such as osteoporosis and osteoarthritis, anddiseases that result in the loss of connective tissue in organs, such ascirrhosis of the liver.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the elution position of substantially pure serum YKL-40 ona gel filtration column.

FIG. 2 depicts the results of a radioimmunoassay for YKL-40 inbiological samples (serum and synorial fluid) taken from human patientswith inflammatory rheumatic joint disease. Purified YKL-40 is indicatedby ●. Serum levels of YKL-40 in a healthy person is indicated by ; inserum from a rheumatoid arthritis patient by _, and in synovial fluid ofa rheumatoid arthritis patient by □.

FIG. 3 depicts the results of assays for levels of YKL-40 and otherbiochemical markers of joint disease in serum taken from human patientsdiagnosed as having joint disease.

FIGS. 4( a)-(b) depict changes in serum YKL-40 and serum hyaluronanlevels in 57 patients with active RA during a one year study periodregarding the effects of methylprednisolone (MP) treatment on RA. Thepatients indicated by ● in the figure received MP treatment, whilepatients in a control group received a placebo (indicated by ∘). Allpatients received either penicillamine or azathioprine.

FIGS. 5( a)-(b) depict changes in serum YKL-40 and serum hyaluronanlevels in 57 patients with active RA during a one year study periodregarding the effects of methylprednisolone (MP) treatment on RA. Thepatients indicated by ● in the figure received MP treatment, whilepatients in a control group received a placebo (indicated by ∘). Allpatients received either penicillamine or azathioprine. For eachsubject, the initial concentration was set at 100%, and all subsequentvalues were expressed as a percentage of each initial value.

FIG. 6 shows a Kaplan-Meier survival curve, which relates the serumlevels of YKL-40 measured in 60 breast cancer patients (aged 29-78years) following recurrence and metastasis of their cancers to thelength of time that each patient subsequently survived.

FIG. 7 depicts levels of YKL-40 detected in the sera and synovial fluidfrom 137 disease-free women, aged 2079 years.

FIG. 8 is a graph which identifies the serum levels of YKL-40 in breastcancer patients (measured as described with respect to FIG. 6) and showsif and when each patient subsequently died as a result of their illness.The data are identified according to the selection criteria for entranceinto the study (described in Example VII) that were met by the patient..circle-solid. or .circle-solid.=patients meeting selection criteria #1;□ or ▪=patients with no recurrence of breast cancer; X=patients meetingselection criteria #2; and, or .Δ=patients meeting selection criteria#3. Open and X symbols denote patients still alive at the point in timenoted; closed symbols denote patients who had died by the time noted.

FIG. 9 depicts YKL-40 levels detected in the sera of patients in a studyregarding recurring, metastatic breast cancer in relation to theprincipal site of metastasis (if any) of the cancer. The data areidentified according to the selection criteria for entrance into thestudy (described in Example VIII) that were met by the patient.=patientsmeeting selection criteria #1; □=patients with no recurrence of breastcancer; X=patients meeting selection criteria #2; and,=patients meetingselection criteria #3.

FIG. 10 depicts YKL-40 levels detected in the sera of patients in astudy regarding recurring breast cancer with metastasis to bone butwithout visceral involvement of the cancer. The data are identifiedaccording to the selection criteria for entrance into the study(described in Example VIII) that were met by the patient..circle-solid.=patients meeting selection criteria #1; □=patients withno recurrence of breast cancer; X=patients meeting selection criteria#2; and,=patients meeting selection criteria #3.

DETAILED DESCRIPTION OF THE INVENTION

A. Definitions

The following definitions are provided to simplify discussion of theinvention. They should not, therefore, be construed as limiting theinvention, which is defined in scope by the appended claims.

“Antibody” includes intact molecules as well as fragments thereof suchas Fab and F(ab′)₂ which are capable of binding an epitopic determinanton the YKL-40 protein.

“Antigen” (as used in the context of the inventive assay) refers to theYKL-40 protein. The N-terminal amino acid sequence of this protein isset forth in the sequence listing herein as SEQ ID. NO: 1.

“Mammal” as used herein includes both humans and non-humans.

“mAb” refers to a monoclonal antibody.

“Substantially pure”, as used to describe YKL-40, refers to thesubstantially intact molecule which is essentially free of othermolecules with which YKL-40 may be found in nature.

“Disease state” refers to an illness or injury in a mammal.

“Associated” with respect to the role in YKL-40 in a disease state in amammal refers to release of YKL-40 into a tissue or fluid of the mammalwhich release occurs during or at the onset of the disease state and isthe result of the onset or occurrence of the disease state.

“Ameliorate” refers to a lessening in the severity of a disease state,including remission or cure thereof.

B. Isolation and Purification of YKL-40.

To develop antibodies for use in all assay procedures and antigen foruse in competitive assay procedures according to the methods of theinvention, YKL-40 must be obtained from a biological sample orsynthesized, preferably in a substantially pure form. Native YKL-40 maybe obtained from any mammalian fluid or tissue in which it is known tobe present. Although the normal distribution of YKL-40 in mammals is asyet not completely known, it has been found in serum, synovial fluid andconditioned media of chondrocytes and osteosarcoma cells (MG63 cellline, American Type Culture Collection, Rockville, Md. [“ATCC”]).Northern blot analyses have shown that YKL-40 mRNA is expressed at highlevels in the liver, weakly by brain, kidney and placenta, and atundetectable levels by heart, lung, and skelatal muscle (Hakala, et al.(1993) J. Biol. Chem, 268: 25803-25810).

Condition media can be prepared by culturing YKL-40 producing cellsaccording to means known in the art, preferably using RPMI 1640serum-free media (Irvine Scientific, Irvine, Calif.). YKL-40 is purifiedaccording to means known in the art, such as by affinity chromatographyor gel filtration (on, for example, the resin SEPHACRYL S-200-HR fromPharmacia, Piscataway, N.J.). YKL-40 has a molecular weight of about 40kD. The N-terminal amino acid sequence is shown in the sequence listingas SEQ ID. NO. 1; the full coding region of the gene for YKL-40 iscontained in SEQ ID NO:4. Substantial homology along the N-terminal andinternal amino sequences (the latter of which are shown in SEQ ID NO. 2,(“YKL-40 peptide A”) and SEQ. ID. NO:3, (“YKL-40 peptide B”)) with abacterial polysaccharide hydrolase (chitinase) supports the conclusionthat YKL-40 degrades polysaccharide components in connective tissue.Specifically, SEQ ID. NO. 2 correlates to 14/19 residues of an internalamino acid sequence for chitinase, while about 50% of the residues inthe N-terminal sequence for YKL-40 correlate to the N-terminus ofchitinase (SEQ. ID. NO. 3). YKL-40 also has substantial sequenceidentity to a protein secreted by activated murine macrophages (PIRAccession No. S27879). Allowing for some gaps in sequence alignment,there are 142 identities between residues 26 to 359 of the complete 383residue sequence of YKL-40 (GenBank Accession No. M80927; see, SEQ IDNO:4), and residues 27-369 of the 505 residue macrophage secretoryprotein.

Although it is not intended that the invention be limited by aparticular theory regarding the mechanism by which YKL-40 functions in agiven disease state, such sequence identities strongly suggest thatYKL-40 is an enzyme that hydrolyzes glycosidic bonds in an as yetunidentified macromolecule in the extracellular environment of cells.Since chitin itself is not found in vertebrates, and since YKL-40apparently does not possess chitinase activity, it is probable thatdivergent evolution of in ancestral chitinase altered the specificity ofthe vertebrate enzyme so that it now cleaves a different glycosidiclinkage than the one targeted by chitinase.

In healthy connective tissue, YKL-40 may play a role in normal tissueremodeling. Given the substantial increase in YKL-40 detected asdescribed below in the sera and synovial fluid of persons afflicted withconnective tissue degradative diseases, and the apparent absence ofYKL-40 in healthy cells, it is believed that the production and/orsecretion of YKL-40 in diseases associated with YKL-40 is upregulated toan abnormal level through an as yet unknown disease process. Thus, it islikely, that YKL-40 is a cellular product which plays an active role inthe disease process rather than merely a structural component ofdegraded connective tissue. For ease of reference, therefore, connectivetissue on which secreted YKL-40 acts or in which it is secreted will bereferred to herein as connective tissue “containing” YKL-40.

For use in the inventive assay, YKL-40 and immunogenic fragments thereofmay also be synthesized according to means which are well-known in theart. Using conventional techniques, the full-length gene can beexpressed using suitable expression vectors known in the art or thepeptide can be chemically constructed using amino acids corresponding tothe deduced amino acid sequence for YKL-40.

For example, YKL-40 may be synthesized without undue experimentation bycommonly used methods such as t-BOC or FMOC protection of alpha-aminogroups. Both methods involve stepwise synthesis whereby a single aminoacid is added at each step starting from the C terminus of the peptide(see, Coligan, et al., Current Protocols in Immunology, WileyInterscience, 991, Unit 9). Peptides of the invention can also besynthesized by various well known solid phase peptide synthesis methods,such as those described by Merrifield, J. Am. Chem. Soc., 85:2149(1962), and Stewart and Young, Solid Phase Peptides Synthesis, (Freeman,San Francisco, 27-62, 1969), using a copoly(styrene-divinylbenzene)containing 0.1-1.0 mMol amines/g polymer.

In this latter method, upon completion of chemical synthesis, thepeptides can be deprotected and cleaved from the polymer by treatmentwith liquid HF-10% anisole for about ¼-1 hours at 0.degree. C. Afterevaporation of the reagents, the peptides are extracted from the polymerwith 1% acetic acid solution which is then lyophilized to yield thecrude material. This can normally be purified by such techniques as gelfiltration on Sephadex G-15 using 5% acetic acid as a solvent.Lyophilization of appropriate fractions of the column will yield thehomogeneous peptide or peptide derivatives, which can then becharacterized by such standard techniques as amino acid analysis, thinlayer chromatography, high performance liquid chromatography,ultraviolet absorption spectroscopy, molar rotation, solubility, andquantitated by the solid phase Edman degradation.

DNA sequences for use in producing YKL-40 and YKL-40 peptides can alsobe obtained by several methods. For example, the DNA can be isolatedusing hybridization procedures which are well known in the art. Theseinclude, but are not limited to: 1) hybridization of probes to genomicor cDNA libraries to detect shared nucleotide sequences; 2) antibodyscreening of expression libraries to detect shared structural featuresand 3) synthesis by the polymerase chain reaction (PCR).

Hybridization procedures are useful for the screening of recombinantclones by using labeled mixed synthetic oligonucleotide probes whereeach probe is potentially the complete complement of a specific DNAsequence in the hybridization sample which includes a heterogeneousmixture of denatured double-stranded DNA. For such screening,hybridization is preferably performed on either single-stranded DNA ordenatured double-stranded DNA. Hybridization is particularly useful inthe detection of cDNA clones derived from sources where an extremely lowamount of mRNA sequences relating to the polypeptide of interest arepresent. In other words, by using stringent hybridization conditions(Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor,1984) directed to avoid non-specific binding, it is possible, forexample, to allow the autoradiographic visualization of a specific cDNAclone by the hybridization of the target DNA to that single probe in themixture.

A YKL-40 containing cDNA library can be screened by injecting thevarious mRNA derived from cDNAs into oocytes, allowing sufficient timefor expression of the cDNA gene products to occur, and testing for thepresence of the desired cDNA expression product, for example, by usingantibody specific for YKL-40 or by using probes for the repeat motifsand a tissue expression pattern characteristic of YKL-40. Alternatively,a cDNA library can be screened indirectly for YKL-40 peptides having atleast one epitope using antibodies specific for the polypeptides. Asdescribed in Section C below, such antibodies can be either polyclonallyor monoclonally derived and used to detect expression product indicativeof the presence of YKL-40 cDNA (see SEQ ID NO:4).

Screening procedures which rely on nucleic acid hybridization make itpossible to isolate any gene sequence from any organism, provided theappropriate probe is available. Oligonucleotide probes, which correspondto a part of the sequence encoding the protein in question, can besynthesized chemically. This requires that short, oligopeptide stretchesof amino acid sequence must be known. The DNA sequence encoding theprotein can be deduced from the genetic code, however, the degeneracy ofthe code must be taken into account. It is possible to perform a mixedaddition reaction when the sequence is degenerate. This includes aheterogeneous mixture of denatured double-stranded DNA. For suchscreening, hybridization is preferably performed on eithersingle-stranded DNA or denatured double-stranded DNA. The development ofspecific DNA sequences encoding YKL-40 or fragments thereof, can also beobtained by: 1) isolation of double-stranded DNA sequences from thegenomic DNA, and 2) chemical manufacture of a DNA sequence to providethe necessary codons for the polypeptide of interest.

The gene encoding YKL-40 may be inserted into a recombinant expressionvector. The term “recombinant expression vector” refers to a plasmid,virus or other vehicle known in the art that has been manipulated byinsertion or incorporation of the appropriate genetic sequences. Suchexpression vectors contain a promoter sequence which facilitates theefficient transcription of the inserted genetic sequence of the host.

Transformation of a host cell with recombinant DNA may also be carriedout by conventional techniques as are well known to those skilled in theart. Where the host is prokaryotic, such as E. coli, competent cellswhich are capable of DNA uptake can be prepared from cells harvestedafter exponential growth phase and subsequently treated by theCaCl.sub.2 method by procedures well known in the art. Alternatively,MgCl.sub.2 or RbCl can be used. Transformation can also be performedafter forming a protoplasm to the host cell or by electroporation.

Isolation and purification of microbially expressed polypeptide, orfragments thereof, provided by the invention, may be carried out byconventional means including preparative chromatography andimmunological separations involving monoclonal or polyclonal antibodies.

Peptides and polynucleotides of the invention include functionalderivatives of YKL-40, YKL-40 peptides and nucleotides encodingtherefor. By “functional derivative” is meant the “fragments,”“variants,” “analogs,” or “chemical derivatives” of a molecule. A“fragment” of a molecule, such as any of the DNA sequences of thepresent invention, includes any nucleotide subset of the molecule. A“variant” of such molecule refers to a naturally occurring moleculesubstantially similar to either the entire molecule, or a fragmentthereof. An “analog” of a molecule refers to a non-natural moleculesubstantially similar to either the entire molecule or a fragmentthereof.

A molecule is said to be “substantially similar” to another molecule ifthe sequence of amino acids in both molecules is substantially the same.Substantially similar amino acid molecules will possess a similarbiological activity. Thus, provided that two molecules possess a similaractivity, they are considered variants as that term is used herein evenif one of the molecules contains additional amino acid residues notfound in the other, or if the sequence of amino acid residues is notidentical.

Further, a molecule is said to be a “chemical derivative” of anothermolecule when it contains additional chemical moieties not normally apart of the molecule. Such moieties may improve the molecule'ssolubility, absorption, biological half-life, etc. The moieties mayalternatively decrease the toxicity of the molecule, eliminate orattenuate any undesirable side effect of the molecule, etc. Moietiescapable of mediating such effects are disclosed, for example, inRemington's Pharmaceutical Sciences, 16th Ed., Mack Publishing Co.,Easton, Pa., 1980.

Minor modifications of the YKL-40 primary amino acid sequence may resultin proteins and peptides that have substantially similar activity ascompared to the YKL-40 peptides described herein. Such modifications maybe deliberate, as by site-directed mutagenesis, or may be spontaneous.All of the peptides produced by these modifications are included hereinas long as the biological activity of YKL-40 still exists. Further,deletion of one or more amino acids can also result in a modification ofthe structure of the resultant molecule without significantly alteringits biological activity. This can lead to the development of a smalleractive molecule which would have broader utility. For example, one canremove amino or carboxy terminal amino acids which may not be requiredfor the enzyme to exert the desired catalytic or antigenic activity.

C. Antibodies to YKL-40.

Either polyclonal or monoclonal antibodies may be used in theimmunoassays and therapeutic methods of the invention described below.Polyclonal antibodies may be raised by multiple subcutaneous orintramuscular injections of substantially pure YKL-40 or antigenicYKL-40 peptides into a suitable non-human mammal. The antigenicity ofYKL-40 peptides can be determined by conventional techniques todetermine the magnitude of the antibody response of an animal which hasbeen immunized with the peptide. Generally, the YKL-40 peptides whichare used to raise the anti-YKL-40 antibodies should generally be thosewhich induce production of high titers of antibody with relatively highaffinity for YKL-40.

If desired, the immunizing peptide may be coupled to a carrier proteinby conjugation using techniques which are well-known in the art. Suchcommonly used carriers which are chemically coupled to the peptideinclude keyhole limpet hemocyanin (KLH), thyroglobulin, bovine serumalbumin (BSA), and tetanus toxoid. The coupled peptide is then used toimmunize the animal (e.g. a mouse or a rabbit). Because YKL-40 may beconserved among mammalian species, use of a carrier protein to enhancethe immunogenecity of YKL-40 proteins is preferred.

The antibodies are then obtained from blood samples taken from themammal. The techniques used to develop polyclonal antibodies are knownin the art see, e.g., Methods of Enzymology, “Production of AntiseraWith Small Doses of Immunogen: Multiple Intradermal Injections”,Langone, et al. eds. (Acad. Press, 1981)). Polyclonal antibodiesproduced by the animals can be further purified, for example, by bindingto and elution from a matrix to which the peptide to which theantibodies were raised is bound. Those of skill in the art will know ofvarious techniques common in the immunology arts for purification and/orconcentration of polyclonal antibodies, as well as monoclonal antibodiesSee, for example, Coligan, et al., Unit 9, Current Protocols inImmunology, Wiley Interscience, 1991).

Preferably, however, the YKL-40 antibodies produced will be monoclonalantibodies (“mAb's”). For preparation of monoclonal antibodies,immunization of a mouse or rat is preferred. The term “antibody” as usedin this invention includes intact molecules as well as fragmentsthereof, such as, Fab and F(ab′).sub.2, which are capable of binding anepitopic determinant. Also, in this context, the term “mAb's of theinvention” refers to monoclonal antibodies with specificity for YKL-40.

The general method used for production of hybridomas secreting mAbs iswell known (Kohler and Milstein, Nature, 256:495, 1975). Briefly, asdescribed by Kohler and Milstein the technique comprised isolatinglymphocytes from regional draining lymph nodes of five separate cancerpatients with either melanoma, teratocarcinoma or cancer of the cervix,glioma or lung, (where samples were obtained from surgical specimens),pooling the cells, and fusing the cells with SHFP-1. Hybridomas werescreened for production of antibody which bound to cancer cell lines.

Confirmation of YKL-40 specificity among mAb's can be accomplished usingrelatively routine screening techniques (such as the enzyme-linkedimmunosorbent assay, or “ELISA”) to determine the elementary reactionpattern of the mAb of interest.

It is also possible to evaluate an mAb to determine whether it has thesame specificity as a mAb of the invention without undue experimentationby determining whether the mAb being tested prevents a mAb of theinvention from binding to YKL-40 isolated as described above, if the mAbbeing tested competes with the mAb of the invention, as shown by adecrease in binding by the mAb of the invention, then it is likely thatthe two monoclonal antibodies bind to the same or a closely relatedepitope.

Still another way to determine whether a mAb has the specificity of amAb of the invention is to pre-incubate the mAb of the invention with anantigen with which it is normally reactive, and determine if the mAbbeing tested is inhibited in its ability to bind the antigen. If the mAbbeing tested is inhibited then, in all likelihood, it has the same, or aclosely related, epitopic specificity as the mAb of the invention.

D. Immunoassay Procedures.

The immunoassay procedure used must be quantitative so that levels ofYKL-40 in a patient with disease may be distinguished from normal levelswhich may be present in healthy humans and/or background levels measuredin the patient. Competitive and sandwich assays on a solid phase usingdetectible labels (direct or indirect) are, therefore, preferred. Thelabel will provide a detectible signal indicative of binding of antibodyto the YKL-40 antigen. The antibody or antigen may be labelled with anylabel known in the art to provide a detectible signal, includingradioisotopes, enzymes, fluorescent molecules, chemiluminescentmolecules, bioluminescent molecules and colloidal gold. Of the knownassay procedures, radioimmunoassay (RIA) is most preferred for itssensitivity. A radioisotope will, therefore, be the preferred label.

Examples of metallic ions which can be directly bound to an antibody, orindirectly bound to the YKL-40 antigen are well-known to those ofordinary skill in the art and include .sup.125 I, .sup.111 In, .sup.97Ru, .sup.67 Ga, .sup.68 Ga, .sup.72 As, .sup.89 Zr, .sup.90 Y and.sup.201 T1. Preferred for its ease of attachment without compromise ofantigen binding specificity is .sup.125 I (sodium salt, Amersham, UnitedKingdom). Labelling of YKL-40 with .sup.125 I may be performed accordingto the method described in Salacinski, et al., Anal. Biochem.,117:136-146, 1981. Iodogen for use to provide the .sup.125 I label(1,3,4,6-tetrachloro-3.alpha., 6.alpha.-diphenyl glycoluril) iscommercially available from Pierce and Warriner, Chester, England.

The radioimmunoassay of the invention uses standards or samplesincubated with a substantially equal volume of YKL-40 antiserum and ofYKL-40 tracer. Standards and samples are generally assayed in duplicate.The sensitivity (detection limit) of the assay of the invention is about10 μg/L. Sensitivity in this context is defined as the detectible massequivalent to twice the standard deviation of the zero binding values.The standard curve will generally be linear between 20 and 100 μg/L Theintra- and interassay coefficients of variance for the assay describedin the following examples are <6.5% and <12%, respectively.

It will be appreciated by those skilled in the art that, although notnecessarily as sensitive as an RIA, assay procedures using labels otherthan radioisotopes have certain advantages and may, therefore, beemployed as alternatives to the preferred RIA format. For example, anenzyme-linked immunosorbent assay (ELISA) may be readily automated usingan ELISA microtiter plate reader and reagents which are readilyavailable in many research and clinical laboratories. Fluorescent,chemiluminescent and bioluminescent labels have the advantage of beingvisually detectible, though they are not as useful as radioisotopes toquantify the amount of antigen bound by antibody in the assay.

Further, it will be appreciated by those of skill in the art that meansother than immunoassays may be employed to detect and quantify thepresence of YKL-40 in a biological sample. For example, a polynucleotideencoding YKL-40 may be detected using quantitative polymerase chainreaction (PCR) protocols known in the art. The preferred method forperformance of quantitative PCR is a competitive PCR technique performedusing a competitor template containing an induced mutation of one ormore base pairs which results in the competitor differing in sequence orsize from the target YKL-40 gene template. One of the primers isbiotinylated or, preferably, aminated so that one strand (usually theantisense strand) of the resulting PCR product can be immobilized via anamino-carboxyl, amino-amino, biotin-streptavidin or other suitably tightbond to a solid phase support which has been tightly bound to anappropriate reactant. Most preferably, the bonds between the PCRproduct, solid phase support and reactant will be covalent ones, thusreliably rendering the bonds resistant to uncoupling under denaturingconditions.

Once the aminated or biotinylated strands of the PCR products areimmobilized, the unbound complementary strands are separated in analkaline denaturing wash and removed from the reaction environment.Sequence-specific oligonucleotides (“SSO's”) corresponding to the targetand competitor nucleic acids are labelled with a detection tag. TheSSO's are then hybridized to the antisense strands in absence ofcompetition from the removed unbound sense strands. Appropriate assayreagents are added and the degree of hybridization is measured by ELISAmeasurement means appropriate to the detection tag and solid phasesupport means used, preferably an ELISA microplate reader. The measuredvalues are compared to derive target nucleic acid content, using astandard curve separately derived from PCR reactions amplifyingtemplates including target and competitor templates. This method isadvantageous in that it is quantitative, does not depend upon the numberof PCR cycles, and is not influenced by competition between the SSOprobe and the complementary strand in the PCR product.

Alternatively, part of the polymerization step and all of thehybridization step can be performed on a solid phase support. In thismethod, it is an nucleotide polymerization primer (preferably anoligonucleotide) which is captured onto a solid phase support ratherthan a strand of the PCR products. Target and competitor nucleic acidPCR products are then added in solution to the solid phase support and apolymerization step is performed. The unbound sense strands of thepolymerization product are removed under the denaturing conditionsdescribed above.

A target to competitor nucleic acid ratio can be determined by detectionof labelled oligonucleotide SSO probes using appropriate measurementmeans (preferably ELISA readers) and standard curve as described supra.The efficiency of this method can be so great that a chain reaction inthe polymerization step may be unnecessary, thus shortening the timeneeded to perform the method. The accuracy of the method is alsoenhanced because the final polymerization products do not have to betransferred from a reaction tube to a solid phase support forhybridization, thus limiting the potential for their loss or damage. Ifnecessary for a particular sample, however, the PCR may be used toamplify the target and competitor nucleic acids in a separate reactiontube, followed by a final polymerization performed on the solid phasesupport.

Molecules capable of providing different, detectible signals indicativeof the formation of bound PCR products known to those skilled in the art(such as labelled nucleotide chromophores which will form differentcolors indicative of the formation of target and competitor PCRproducts) can be added to the reaction solution during the last fewcycles of the reaction. The ratio between the target and competitornucleic acids can also be determined by ELISA or other appropriatemeasurement means and reagents reactive with detection tags coupled tothe 3′ end of the immobilized hybridization primers. This method mayalso be adapted to detect whether a particular gene is present in thesample (without quantifying it) by performing a conventionalnoncompetitive PCR protocol.

Those of ordinary skill in the art will know, or may readily ascertain,how to select suitable primers for use in the above methods. For furtherdetails regarding the above-described techniques, reference may be madeto the disclosures in Kohsaka, et al., Nuc. Acids Res., 21:3469-3472,1993; Bunn, et al., U.S. Pat. No. 5,213,961; and to Innis, et al., PCRProtocols: A Guide to Methods and Applications, Acad. Press, 1990, thedisclosures of which are incorporated herein solely for purposes ofillustrating the state of the art regarding quanititative PCR protocols.

E. Diagnostic Application.

As shown in examples provided below, diagnosis of disease based onmeasured levels of YKL-40 can be made by comparison to levels measuredin a disease-free control group or background levels measured in aparticular patient. The diagnosis can be confirmed by correlation of theassay results with other signs of disease known to those skilled in theclinical arts, such as the diagnostic standards for RA and breast cancerdescribed in the examples below.

Where the amelioration of a disease (such as RA) can be related toreduction in levels of YKL-40 (and concomitant cartilage repair), YKL-40levels in a biological assay sample taken from the patient (e.g.,synovial fluid) should be measured before (for background) andperiodically during the course of treatment. Because reductions inYKL-40 levels may be transient, the assay will preferably be performedat regular intervals (e.g., every 4 weeks) closely before and after eachtreatment. Depending on the course of treatment, tumor load and otherclinical variables, clinicians of ordinary skill in the art will be ableto determine an appropriate schedule for performing the assay fordiagnostic or disease/treatment monitoring purposes.

Because in certain instances serum YKL-40 may stem from sources otherthan the tissue of interest, a sample should, if possible, be taken fromthe tissue of interest. For example, for diagnosis or monitoring ofjoint disease the assay sample will preferably be drawn from thesynovial fluid of the affected or potentially affected joint. Fordiagnosis and monitoring of tumor metastasis, however, the preferredsource for the assay sample will be blood. Those of ordinary skill inthe art will be able to readily determine which assay sample source ismost appropriate for use in diagnosis of a particular disease for whichYKL-40 is a marker.

The levels of YKL-40 which are indicative of the development oramelioration of a particular disease will vary by disease and, to alesser extent, by patient. Generally, however, as demonstrated by thedata presented in the Examples, the median concentration of YKL-40detected in sera from a sample group of 736 children and adults was 80μg/l in children (aged 6-17 years) and 102 μg/l in adults (aged 20-79years). No statistically significant variations between these valueswere observed between different age groups of children or adults youngerthan 69 years. Adults older than 69 years, however, tended toward higherserum YKL-40 levels than were present in the sera of adults younger than69 years. Thus, for purposes of diagnosing the onset, progression, oramelioration of disease, variations in the levels of YKL-40 of interestwill be those which differ on a statistically significant level from thenormal (i.e., healthy) population and which correlate to other clinicalsigns of disease occurrence and/or amelioration known to those skilledin the clinical art pertaining to the disease of interest (i.e.“diagnostically significant levels” of YKL-40).

For example, in inflammatory joint diseases synovial fluid YKL-40 levelscan be correlated to other biochemical markers of joint disease, inparticular elastolytic activity by monocytes and macrophages for thedegradation of proteoglycans and collagens in synovial fluid. YKL-40levels also correlate well to elevated IL-6 levels in synovial fluid.IL-6 is secreted by chondrocytes and synovial cells and serves toregulate immune responses, including inflammation. Relatively highlevels of IL-6 are found in the synovial fluid of patients withinflammatory and degenerative arthropathies.

Correlation also exists to a somewhat lesser extent between YKL-40levels and acute C-reactive protein (CRP) levels. CRP is present inelevated quantities in the acute phase of rheumatic joint diseases andappears to play a biologic role in inflammation. YKL-40 levels similarlycorrelate with serum P-III-NP levels, which reflect local inflammatoryalterations in type III collagen metabolism in synovial fluid. Althoughit is not intended that the invention be limited to a particulardiagnosis, the correlation of YKL-40 levels suggests that its levels mayin particular be indicative of inflammation in joint disease. Of course,any diagnosis indicated by YKL-40 measurements made according to themethods of the invention will be independently confirmed with referenceto clinical manifestations of disease known to practitioners of ordinaryskill in the clinical arts.

By way of further example, in breast cancer patients, serum YKL-40levels are elevated in patients with cancer cell metastasis as comparedto patients without breast cancer. It is probable that the elevatedlevels of YKL-40 in serum are produced at least in part by degenerationof the connective barrier to the entrance of cancer cells into blood. Itcan be expected that a similar process may accompany entrance of cancercells into lymphatic circulation. As demonstrated by the data presentedbelow, the detected elevations in serum YKL-40 appear to be indicativeof metastasis to viscera and bone, rather than to localised sites, skinor solitary lymph glands. However, the latter metastases may be detectedfairly readily by conventional medical examination.

Further, greatly elevated levels of YKL-40 appear in the sera ofpatients who have experienced a metastatic recurrence of breast cancer(in particular, with metastasis to bone and/or viscera). As compared toa median concentration of serum YKL-40 in age-matched controls (about102 μg/1), patients with confirmed metastases to bone (the most commonsite of breast cancer cell metastasis) had a median concentration ofserum YKL-40 of about 328 μg/l. Further, patients with confirmedmetastases to viscera had a median concentration of serum YKL-40 ofabout 157 μg/l.

In contrast, markers now in common use for bone metastases (serum totalalkaline phosphatase, bone alkaline phophatase and bone Gla protein)show considerable variation in patients with metastatic breast cancer;increases in serum bone Gla protein in particular have not been shown tobe diagnostic for breast cancer metastasis to bone.

Interestingly, elevation of serum levels of YKL-40 correlate to thenumber of months each patient can be expected to survive followingrecurrence of the cancer, particularly in those patients having serumYKL-40 levels equal to or greater than about 164 μg/l, most particularlyin those patients having serum YKL-40 levels equal to or greater thanabout 207 μg/l (i.e., “prognostically significant levels” of YKL-40).Generally, the higher the level of YKL-40, the shorter the period ofsurvival.

F. Drug Screening Application

As discussed above, YKL-40 appears to be a hydrolytic enzyme whoseproduction is increased during the course of a disease state associatedwith the degeneration of connective tissue. In particular, it appearsthat YKL-40 is involved in the digestion of connective tissue thatcauses the loss of such tissue when it is digested more rapidly than itis repaired. Logically, therefore, agents that inhibit the productionand/or activity of YKL-40 would be expected to limit connective tissueloss.

To that end, the YKL-40 protein, peptides and antibodies of theinvention will be useful in screening potential inhibitors of YKL-40.Potential inhibitors of YKL-40 activity include substrate molecules thatwill competitively bind to YKL-40 and antibodies specific for YKL-40.For example, potential YKL-40 substrate molecules may be screened andidentified using the substantially pure YKL-40 of the invention in acompetitive immunoassay with YKL-40 antibodies. Those of skill in theart will recognize, however, that substrate molecule binding to YKL-40may also be characterized by determination of other parameters, such asbinding kinetics and affinity.

Once a molecule has been determined to bind YKL-40, other potentialsubstrate molecules may be screened for binding by inhibition and/orcompetitive binding studies as described supra with respect to screeningof mAb's with specificity for YKL-40.

G. Therapeutic Application

Assuming the accuracy of the above prediction of YKL-40 enzyme activity,it can be expected that YKL-40 substrate molecule and anti-YKL-40antibody compositions will have therapeutic efficacy. More specifically,it is expected that YKL-40 activity can be attenuated (thus reducing thehost's response to response to YKL-40; e.g. digestion of connectivetissue) by blocking binding of native YKL-40 substrate to YKL-40 withanti-YKL-40 antibodies and/or by competitive binding of YKL-40 topharmaceutically acceptable substrate molecules.

To that end, YKL-40 substrate molecule or anti-YKL-40 compositions areprepared for administration by mixing YKL-40 substrate molecules havingthe desired degree of purity, or anti-YKL-40 antibodies having thedesired degree of affinity for YKL-40, with physiologically acceptablecarriers. Such carriers will be nontoxic to recipients at the dosagesand concentrations employed. Ordinarily, the preparation of suchcompositions entails combining the particular protein with buffers,antioxidants such as ascorbic acid, low molecular weight (less thanabout 10 residues) polypeptides, proteins, amino acids, carbohydratesincluding glucose or dextrins, chelating agents such as EDTA,glutathione and other stabilizers and excipients. Such compositions mayalso be lyophilized and will be pharmaceutically acceptable; i.e.,suitably prepared and approved for use in the desired application.

For treatment of joint disease and degenerative organ disease (e.g.,cirrohsis of the liver), YKL-40 activity will preferably be targeted inthe joint or organ rather than systemically. Routes of administrationfor the joint or organ of interest (e.g., injection, catheterization)are known to those of ordinary skill in the clinical arts.Alternatively, administration may be by any enternal or parenteral routein dosages which will be varied by the skilled clinician depending anthe patient's presenting condition and the therapeutic ends to beachieved.

The level of YKL-40 activity and/or production may be monitored by theassay described hereinabove as well as by reference to a reduction inclinical manifestations of connective tissue loss associated with thedisease state to be treated. A dosage which achieves this result will beconsidered a “therapeutically effective” dosage. Generally, however,dosages of the YKL-40 substrate molecule will vary from about 10units/m.sup.2 to 20,000 units/m.sup.2, preferably from about 5000 to6000 units/m.sup.2, in one or more dose administrations weekly, for oneor several days.

H. Kits for Use in Therapeutic and Diagnostic Applications.

For use in the diagnostic research and therapeutic applicationssuggested above, kits are also provided by the invention. In thediagnostic and research applications such kits may include any or all ofthe following: assay reagents, buffers, YKL-40 protein and/or fragments,YKL-40 recombinant expression vectors, YKL-40 oligonucleotides and otherhybridization probes and/or primers, YKL-40 substrate molecules and/or asuitable assay device. A therapeutic product may include sterile salineor another pharmaceutically acceptable emulsion and suspension base foruse in reconstituting lyophilized YKL-40 substrate molecules oranti-YKL-40 suspensions, suitably labeled and approved containers ofYKL-40 substrate molecules or anti-YKL-40 compositions, and kitscontaining these products for use in connection with the diagnostic kitcomponents as described above.

Examples illustrating the correlation of YKL-40 levels to joint diseaseactivity, progress of treatment for joint disease, organ degradation,cancer cell metastasis and cancer survival rates are provided below.These examples should not, however, be considered to limit the scope ofthe invention, which is defined by the appended claims.

In the examples, the abbreviation “min.” refers to minutes, “hrs” and“h” refer to hours, and measurement units (such as “ml”) are referred toby standard abbreviations.

EXAMPLE I Isolation and Purification of YKL-40 from Human OsteosarcomaCell Line MG63

YKL-40 was purified from serum-free conditioned medium of the humanosteosarcoma cell line MG63 (MG63 cells were obtained from the AmericanType Culture Collection, Rockville, Md.). Cells were cultured in 100 mmdishes with RPMI 1640 medium containing 10% newborn calf serum, 100Units/ml penicillin, 100 μg/ml streptomycin, 50 μg/ml vitamin C, and 1μg/ml vitamin K.sub.1. The cultures were incubated at 37.degree. C. in ahumidified atmosphere of 10% CO.sub.2. When the cells reachedconfluence, the culture medium was removed and the cell layer was washedtwice with 10 milliliters (ml) of phosphate buffered saline.

Ten mls of serum-free RPMI 1640 media containing 50 μg/ml vitamin C and1 μg/ml vitamin K₁ was then added to each dish. 48 hours later,conditioned medium was decanted from each dish and replaced with 10 mlof fresh serum-free medium containing the same level of addedconstituents. This procedure was repeated every 48 hours for up to 10days. Conditioned medium was freed of cells and debris by centrifugationand stored at −20° C. until use.

YKL-40 was purified by a modification of the heparin-affinitychromatography method described in Nyirkos, et al., (1990) Biochem. J.,269:265-268. Specifically, YKL-40 was first concentrated from 4.75 L ofconditioned medium by adsorption of 40 ml (packed volume) ofHEPARIN-SEPHAROSE CL-68 resin (from Pharmacia) by stirring or 2 hours atroom temperature. The resin was then placed into a 2.times.24 cm columnand washed with 3 column volumes of 0.01 Molar sodium phosphate buffer(pH 7.4) containing 0.05 M NaCl. YKL-40 was eluted from the resin atroom temperature by a linear gradient from 0.05 to 1.2 M NaCl in 0.01Molar sodium phosphate buffer pH 7.4 (200 ml each condition).

To characterize the purity of YKL-40, 5 μl from every third fraction ofthe peak fractions from the HEPARIN-SEPHAROSE CL-6B affinitychromatography procedure described were combined with 25 μl SDS loadingbuffer electrophoresed on a 5-20% SDS-polyacrylamide gradient gel(BioRad, Laboratories, Richmond, Calif.), and stained with Coomassiebrilliant blue. The concentration of the final YKL-40 used for standardand tracer in the inventive assay is based on an absorbance of 1.44 fora 1 milligram (mg) per ml solution of YKL-40.

Articular cartilage was obtained from the knees of cadavers within 18hours of death and of a patient undergoing joint replacement forosteoarthritis, and chondrocytes were isolated by sequential enzymaticdigestion according to methods known in the art (see, e.g., Guerne, etal. (1990) J. Immun., 144:499-505). The resulting cells were ahomogenous population of chondrocytes, since only the superficial layersof cartilage were used for isolation of the cells and, in contrast tofibroblasts or synoviocytes, the cells were nonadherent. The cells werecultured in DMEM-high glucose medium supplemented with 10% fetal calfserum, 100 Units/ml of penicillin, 100 μg/ml streptomycin, and 50 μg/mlvitamin C (Irvine Scientific, Irvine, Calif.). Cells were grown in 175cm.sup.2 tissue culture flasks (primary cultures) or in 100 mm dishes(later passages) in a humidified atmosphere of 10% CO₂ at 37° C. Thecells were subcultured at a 1:3 ratio after trypsinization of confluentmonolayers. To obtain conditioned medium for analysis, the culturemedium was removed after the cells reached confluence and the cell layerwas washed twice with 30 ml (175 cm.sup.2 flasks) or 10 ml (100 mmdishes) of phosphate buffered saline (PBS). The same volume ofserum-free DMEM-high glucose medium containing antibiotics, and 50 μg/mlvitamin C was then added to each culture. Conditioned medium was removedafter 48 hours and replaced with the same volume of fresh serum-freemedium. This procedure was repeated every 48 hours for up to 14 days.Conditioned medium was freed of cells and debris by centrifugation for 5minutes at 1600 g and frozen at −20° C. until use.

EXAMPLE II Preparation of Assay Samples for Radioimmunoassay

1. Assay Sample Sources

Assay samples were obtained from the sera of 49 patients withinflammatory or degenerative joint diseases (34 women and 15 men, aged23-80 years with a median age of 65 years). 29 patients had RA, 7 hadosteoarthritis, 4 had crystal arthritis, 2 had psoriatic arthritis, 5had reactive arthritis and 2 had monoarthritis. Diagnoses were based onthe criteria described in Arnett, et al. (1988) Arthritis Rheum. 31:315-324 (American Rheumatism Association Standards), clinical andradiographic examinations of the knees, and direct microscopy ofsynovial fluid. The patients had a serum CRP level of 25-1600 (median165). 34 patients were taking non-steroidal anti-inflammatory drugs and17 were receiving slow acting antirheumatic agents. 15 patients hadreceived glucocorticoid therapy systemically or locally within the past3 months. The inflammation of the knee was evaluated by a clinical indexrating from 0-6, consisting of palpable synovial swelling (range 0-3)and pain on palpation (0-3).

2. Collection of Serum and Synovial Fluid

Blood samples were allowed to clot at room temperature and thencentrifuged at 1500 g for 10 minutes. Knee joint aspirations wereperformed using conventional aseptic technique without local anesthesia.The synovial fluid was withdrawn from each subject as completely aspossible using a 1.2-mm-gauge needle, and collected in sterile tubescontaining ethylene-diamine-tetracetate (EDTA, 5 mM finalconcentration). The synovial fluid samples were centrifuged at 1800 gfor 30 minutes in order to remove any extraneous debris. The sampleswere either analyzed immediately or stored at −80° C. for lateranalysis.

EXAMPLE III Preparation of Labelled Antigen and Antibodies forRadioimmunoassay for YKL-40

1. Preparation of Radioiodinated YKL-40

Purified YKL-40 was labelled with ¹²⁵I (sodium salt, Amersham, UK)according to the Iodogen method referenced supra. Specifically, 10 μgYKL-40 was incubated for 10 minutes with 18.5 MBq ¹²⁵I using 2 μg ofiodogen (Pierce and Warriner, Chester, England, UK) as oxidant in areaction volume of 110 μl. Iodination was terminated by moving thereaction mixture from the iodogen tube. The labelled YKL-40 wasseparated from free iodine by gel filtration using a SEPHADEX G-25column (1.times.12.5 cm, from Pharmacia) equilibrated with assay buffer(16 mM sodium phosphate buffer pH 7.4, 0.12 M NaCl, 0.1% (w/v) humanserum albumin). The calculated specific activity of the labeled wasabout 15 Ci/g. The elution position of YKL-40 (purified) and of YKL-40taken from the serum of a patient with RA is shown in FIG. 1.

2. Preparation of Antibodies

New Zealand white rabbits were immunized by monthly multiple sitesubcutaneous or intramuscular injection of purified YKL-40. Eachinjection was made with 0.5 mg of human YKL-40 emulsified in incompleteFreund's adjuvant (1:1). The first 4 injections were given at intervalsof two weeks and rabbits were bled 10-12 days after the fourthinjection. Injections were thereafter given at 4 week intervals and theanimals were bled 10-12 days after each injection. Crossedimmunoelectrophoresis showed that the antibodies were monospecific forYKL-40.

It will be understood by those skilled in the art that the radioisotopiclabel could be attached to the antibodies described above rather thanthe antigen with functional equivalence in the assay claimed.

EXAMPLE IV Radioimmunoassay for YKL-40 to Detect YKL-40 Levels in Serumand Synovia of Patients with Rheumatoid Arthritis or Other Joint Disease

The assay samples described in Example II were assayed as follows.YKL-40 antibodies, standards and the tracer were diluted in assaybuffer. In the assay 100 μl of standards or samples were incubated with100 μl of YKL-40 antiserum (1:10,000) and 100 μl of YKL-40 tracer (about15,000 counts/minute) in a final volume of 400 μl at room temperaturefor 20-24 hours. The antibody-bound tracer was then separated byincubation with 100 μl of SAC-CEL (donkey anti-rabbit antibody coatedcellulose suspension; Wellcome Diagnostics Ltd, UK) at room temperaturefor 30 minutes. After addition of 1 ml of distilled water the tubes werecentrifuged at 2000 g for 10 minutes, the supernatant decanted, and theradioactivity of the precipitate counted in an automatic gamma counter(LKB Wallace, CLINIGAMMA 1272) for the time of 10,000 counts.

The precision (intra-assay variation) was calculated from replicatedeterminations (20 times) on each of three quality control sera in asingle assay. The reproductibility (inter-assay variation) wascalculated from data obtained during a 5 month period (20 assays) oneach of three quality control sera. YKL-40 concentrations incorresponding serum and EDTA plasma samples were compared in 75 blooddonors.

All standards and samples were assayed in duplicate. The standard curvewas constructed by use of a spline function.

The individual serum YKL-40 concentrations in the two patient groups andcontrols are shown in FIG. 2. The serum YKL-40 concentrations ofpatients with inflammatory rheumatic disease (median; lowerquartile-upper quartile: 138 μg/L; 103-211 μg/l) was not statisticallydifferent (p=0.44) from those in patients with osteoarthritis (112 μg/L;93-152 μg/L). Serum YKL-40 in both patient groups was significantlyhigher (p<0.001) than that of controls (50 μg/L; 36-64 μg/l). The YKL-40concentration in knee joint synovial fluid from the patients withinflammatory rheumatic disease (2210 μg/l; 1625-3040 μg/l) was notsignificantly different from the concentration of the patients withosteoarthritis (1720 μg/l; 1270-1950 μg/l).

Serum levels of YKL-40 can, therefore be related to the incidence ofjoint disease, particularly inflammatory joint disease. However,distinctions between the different joint diseases evaluated are notapparent from these data.

EXAMPLE V YKL-40 Stability in Serum Assay Samples

To assess the effect of freezing and thawing on YKL-40 antigen in theassay samples, a fresh serum sample was obtained from 6 adults and 10aliquots of each sample were prepared. One aliquot was kept on ice, andthe others were frozen at −20.degree. C. At 60 minute intervals, thealiquots were removed and thawed at room temperature. One sample waskept on ice and the rest refrozen. This procedure was repeated 9 timeswith no loss of serum YKL-40 reactivity. To assess the effect oflong-term storage at room temperature, a fresh serum sample was obtainedfrom 12 adults, and 4 aliquots of each sample were prepared. One aliquotwas immediately frozen at −20.degree. C., the others were frozen after24 hours, 48 hours and 120 hours storage at room temperature, duringwhich time reactivity remained stable.

EXAMPLE VI Correlation Between YKL-40 in Serum and Synovial Fluid andOther Biochemical Markers of Inflammation and Cartilage Remodeling

As shown in FIG. 3, the YKL-40 concentrations measured in the serum andsynovial fluid samples described in Example II were highly correlatedand the synovial fluid/serum YKL-40 ratio was high. YKL-40 levels inserum and synovial fluid correlated significantly with serum CRP,synovial fluid IL-6, and synovial fluid M.o slashed. elastolysis levels,which were also measured by assay of these samples as described below.Serum YKL-40 also correlated with serum M.o slashed. elastolysis andserum P-III-NP levels in these samples. The synovial fluid YKL-40concentrations measured in these samples correlated with a clinicalindex of knee inflammation. No correlation was found between YKL-40 inserum or synovial fluid and serum IL-6 and synovial fluid P-III-NPlevels.

The serum concentration of CRP was determined by nephelometry(Behringwerke, Marburg, Germany). Interleukin-6 (IL-6) activity wasdetermined by bioassay using the highly specific IL-6 dependant mousehybridoma cell line B13, 29 clone B9 known in the art. The aminoterminalpropeptide of type III procollagen (P-III-NP) was measured by acommercially available RIA (P-III-NP RIA kit, Farmos Diagnostica,Oulunsalo, Finland). The elastolytic activity of monocytesimacrophages(M.o slashed.) were investigated with an assay for live M.o slashed.elastolysis described by Jensen, et al. (1991) Scand. J. Rheum.20:83-90. The results of these assays are shown in Table I, below.

TABLE I Relationship between serum and synovial fluid concentrations ofykl-40 and other biochemical markers of rheumatoid arthritis SynovialFluid Serum YKL-40 YKL-40 Serum CRP 0.33* 0.31* Serum IL-6 0.26 −0.10Synovial fluid IL-6 0.60** 0.47* Serum M. phi. elastolysis 0.55** 0.58**Synovial fluid M. phi. elastolysis 0.64** 0.58** Serum PIIINP 0.49 0.13Synovial fluid PIIINP 0.02 −0.23 Clinical Knee Index 0.27 0.34* *p <0.05; **p < 0.01; ***p < 0.001.

EXAMPLE VII Correlation of Changes in Serum YKL-40 Levels with Progressin the Treatment of Joint Disease

This one year, double blind placebo controlled study was primarilyconducted in order to evaluate whether (1) YKL-40 relects diseaseactivity in a large group of patients with active rheumatoid arthritis,and, (2) monthly treatment with intravenous methylprednisolone (MP;known to reduce inflammation for 4-8 weeks after intravenousadministration) enhanced or accelerated the effect of the diseasemodifying drugs penicillamine or azathioprine, using YKL-40 as a markerfor disease progression and/or amelioration. The study included 97patients with definite or classic rheumatoid arthritis (RA) as definedby the American Rheumatism Association (see Arnell, et al., ArthritisRheum., supra).

Blood samples were collected in the morning just before each pulsetreatment and plasma YKL-40 was determined by RIA as described inExample IV. The initial median concentration of serum YKL-40 detected inthe 97 patients with RA was 174 μg/l (range 40-583 μg/l). Plasma YKL-40levels in the RA patients were significantly higher (p.gtoreq.0.001)than in 260 healthy adults whose plasma YKL-40 levels were also tested(102 μg/l median of a range of 38-514 μg/l). For comparison, serumhyaluronan and serum C-reactive protein (CRP) levels were also tested inall of the RA patients and in 99 healthy adults (using a radiometricassay for hyaluronan from Pharmacia, Uppsala, Sweden).

Radiography of the hands, wrists and feet of each patient was takenbefore the start of treatment and again at 360 days. The radiographswere evaluated blind by a radiologist. The presence of erosions at least1 millimeter deep and any increase or change in the number of erosionsafter 12 months were noted for clinical significance.

In Table II, the clinical data and initial values of serum YKL-40 andserum hyaluronan in the two treatment groups are shown. The groups werenot significantly different.

TABLE II Clinical Data and Initial Values of Plasma YKL-40 and SerumHyaluronan in the Two Treatment Groups MethylprednisoloneMethylprednisolone Group Placebo Group N = 31 N = 26 Sex (M/F) ratio11/20  4/22 Penicillamine/Azathioprine ratio 20/11 18/8  Age Years 60(23–79) 62.5 (32–78) Disease duration years 9 (1–32) 7.5 (0–43) ERRmm/hour 45 (6–118) 54 (2–110) Serum CRP mg/L 23 (1–248) 42 (1–134)Plasma YKL-40 μg/L 179 (40–583) 185 (44–583) Serum Hyaluronan μg/L 93(14–1196) 121 (35–632) # Bone erosions 15 (0–35) 11 (0–30) # Swollenjoints 7 (0–19) 8 (0–30) # Tender joints 17 (2–45) 22 (2–41) Values aremedians (range). The groups were not significantly different in any ofthe parameters. ESR = erythrocyte sedimentation rate; CRP = C-reactiveprotein; # = Number.

The patients entered a double blind placebo controlled trial of pulsetreatment with 1000 mg intravenously injected methylprednisolone (MP)every 4 weeks for a total of six (6) times, followed by six monthswithout pulse therapy. 7 days after the first pulse therapy the patientswere started on penicillamine or azathioprine as described in Hansen, etal. (1990) Br. Med. J., 301: 268. 57 patients completed the trial,taking the same disease modifying drug throughout (31 were treated withMP [penicillamine/azathioprine: 20:11] and 26 with placebo). Elevenpatients changed from penicillamine to azathioprine during the study,and 29 were withdrawn from the study owing to adverse reactions or lackof effect during treatment with azathioprine.

Data obtained from the study were analyzed statistically as follows. Inorder to evaluate the longitudinal changes in serum YKL-40 and serumhyaluronan during treatment, the initial value for each subject was setat 100% and values obtained after institution of treatment wereexpressed as a percentage of the initial value. The mean differencesbetween groups were evaluated by the Student's t-test for unpaired data.When raw YKL-40 value data was evaluated, comparison between groups werecalculated using the non-parametric mann-Whitney test for unpaireddifferences and the Wilconxon test for paired differences. For theseanalyses, p values of less than 0.05 were considered significant.Correlations between the different parameters were calculated using theSpearman rho test. For this analysis p values of less than 0.01 wereconsidered to be significant. All analytical tests used are standard inthe art.

FIG. 4( a) illustrates the changes in serum YKL-40 and serum hyaluronanduring the one year study period in the 57 patients who completed thetrial. YKL-40 levels were significantly lower compared to initial valuesafter MP therapy (o in FIG. 4( a)) was instituted, then returned toinitial values after therapy was withdrawn. In contrast, in the grouptreated only with penicillamine or azathioprine (.smallcircle. in FIG.4( a)), YKL-40 levels only differed significantly from background afterday 84.

The effects of MP treatment on YKL-40 levels were also compared to itseffect on other biochemical markers of joint disease. Serum hyaluronanwas determined by a radiometric assay using specific hyaluronan-bindingprotein isolated from bovine cartilage (Pharmacia, Uppsala, Sweden).Serum C reactive protein (CRP) was determined by nephelometry(Beringwerke, Marburg, Germany).

Serum hyaluronan was only significantly decreased in the MP group at day1 and day 168 (.circle-solid. in FIG. 4( b); the placebo group isindicated by a .smallcircle.), whereas serum CRP was significantlydecreased throughout the study period in the MP group and after day 84in the placebo group (data not shown). The values measured forhyaluronan did not significantly differ between the MP and placebogroups except at day 1 (see, FIG. 4( b)).

The changes in plasma YKL-40 during the course of the MP treatment wasdifferent compared to the changes in serum hyaluronan, as shown in FIGS.5( a) and (b).

MP therapy therefore produced a significant, albeit transient decreasein plasma YKL-40 which correlated in the first months followingtreatment to other indicators of a therapeutic response.

EXAMPLE VIII Relationship of Serum YKL-40 Levels to Survival RatesFollowing Recurrence of Breast Cancer

Serum levels of YKL-40 were measured in a clinical group of 60 breastcancer patients (aged 29-78 years) using the RIA described in ExampleIV. For comparison, serum YKL-40 levels in a control group of 137disease-free women (aged 20-79 years) were also measured. These lattermeasurements define the normal and median YKL-40 values referred to inthis example.

The members of the clinical and control groups were, respectively:

1. Clinical Group

60 women aged 29-78 years who had previously been diagnosed with primarybreast cancer. They were all potential candidates for systemicantineoplastic treatment. The criteria of entry were: 1) suspicion ofdistant metastases after primary treatment of localized disease; 2)locally advanced disease or distant metastases at the time of initialdiagnosis; and 3) patients with suspected progression of bone metastasisafter initial recurrence. Patients who had other primary cancers at anytime were not eligible for this study.

39 patients (65%) had received adjuvant therapy. 22 (56%) of thesepatients had received adjuvant combination chemotherapy withcyclophosphamide, methotrexate and 5-fluorouracil immediately after theremoval of the primary tumor. None of the patients had been treatedduring the previous 12 weeks before the start of the study (i.e., thetime of assay sample collection).

2. Control Group

Serum YKL-40 concentrations in 137 healthy women (aged 20-79 years) wereestablished for use as control values. The serum samples were obtainedfrom blood donors who attended the Regional Blood Transfusion Servicesat Hvidovre Hospital, Denmark, from women working at different museumsin Copenhagen, Denmark and from elderly women living in a shared housefor elderly in Copenhagen. All these women were healthy (had no knowndisease), were not taking any medicine and all had a normal liver andkidney function.

The period of time which each patient in the clinical group survivedfollowing recurrence of their cancer was observed. The nature of anymetastasis of the tumor cells was also characterized in each patient.These data are correlated to the serum YKL-40 levels measured in eachpatient at the time of recurrence of their cancer.

3. Recurrence

All serum analyses reported here were determined on blood samplesobtained from each of 60 women at the time of their entrance into thestudy. Forty-seven of these women entered the study at the time thatbreast cancer recurrence was first suspected (criteria 1). Further testsrevealed that 6 of these women did not in fact have breast cancerrecurrence. Six women entered the study because they had locallyadvanced disease or distant metastases at the time of initial breastcancer diagnosis (criteria 2) and 7 women entered the study because theywere suspected to have bone metastases 9 to 27 months after their firstrecurrence of breast cancer (criteria 3).

4. Survival After Recurrence.

At the time of analysis 9 of the 60 patients were still alive. Themedian survival after recurrence in the 41 patients with firstrecurrence of breast cancer was 16 months (25-75% fractiles: 9-26months) and in all 60 patients the corresponding values were 16 months(7-40) months). Table III summarizes the univariate survival data for 17variables. Age, degree of anaplasia, serum LDH, serum AP, serum albuminand serum YKL-40 were all significant univariate prognostic factors inthe 60 patients. FIG. 8 shows the individual serum YKL-40 concentrationin relation to months of survival after recurrence. At the time offollow-up all 25 patients with high serum YKL-40 were dead compared to26 of 35 patients with normal serum YKL-40. Sixty-seven percent (20/30)of the patients who died within 16 months had elevated serum YKL-40.

The Kaplan-Meier survival curves according to serum YKL-40 levels in the41 patients with first recurrence of breast cancer are presented in FIG.6. Although the number is small, the survival of the two groups(patients with normal or high serum YKL-40) is explicitly different. Inthe 41 patients with first recurrence of breast cancer the survivalrates after 18 months were 60% for patients with normal and 24% forpatients with high serum YKL-40 (p<0.0009) If the calculations wereperformed on all 60 patients the survival rates after 18 months were 63%and 20% for patients with normal and high levels of serum YKL-40(p<0.0001).

As shown in FIG. 6, 76% of the clinical group members still alive after16 months following recurrence had serum YKL-40 levels of 164 μg/L orless. 85% of the members who lived longer than 30 months followingrecurrence had serum YKL-40 levels of 164 μg/L or less. Thus, patientsurvival after the first recurrence of the cancer was significantlyprolonged (p=0.0009) in the group of patients with normal serum YKL-40compared to the patients with serum YKL-40 levels equal to or greaterthan about 164 μg/l, and particularly in those patients with serumYKL-40 levels equal to or greater than about 207 μg/l (“prognosticallysignificant levels” of YKL-40). These data indicate that an elevatedserum YKL-40 level correlates to decreased survival of patients withadvanced breast cancer, thus suggesting that where such levels aredetected, more aggressive treatment protocols may be warranted. SerumYKL-40 measurements will be especially informative where, as was thecase among the patients in this study, the clinical symptoms of patientswho died more quickly did not differ substantially from the clinicalsymptoms of patients who survived for longer periods followingrecurrence of their cancers.

Similar Kaplain-Meier curves based on serum levels of other bloodproteins (such as serum alkaline phosphatase) measured at the same timeas the YKL-40 levels did not correlate as clearly to survival rate amongthe clinical group members.

5. Location of Metastases.

Among the 60 women in the study, thirty patients (50%) had soft tissuerecurrence; bone metastases (as detected by X-ray or bone biopsy) werefound in 40 patients (67%); and visceral metastases (lung, pleura orliver) occurred in 19 (32%) patients.

FIG. 9 shows the distribution of serum YKL-40 according to main sites ofmetastases among the patients in the clinical group. All six patientswithout metastases had a normal serum YKL-40 level. The Kruskal-Wallistest of the YKL-40 levels between the groups was highly significant(p=0.03). The median serum YKL-40 in patients with visceral or bonemetastases was significantly higher (p<05) compared to the levels inpatients without metastases and to the level in healthy age-matchedwomen (102 μg/l, p<0.001). If only the 41 patients with first recurrenceof breast cancer were used in the calculations similar significance ofdifference were found.

Twenty-five of the 54 patients with metastases had serum YKL-40 levelsabove the cut-off level of 207 μg/l. In patients with soft tissuerecurrence (n=10), the median serum YKL-40 was 123 μg/L, and only 2patients had elevated serum YKL-40. One of these 2 patients had a veryhigh serum YKL-40 concentration (904 μg/l) and died after 5 months. Atthe time of blood sampling, this patient had pleuraeffusion butmicroscopy did not reveal malignant cells. In patients with bonemetastases (=/-soft tissue recurrence (N=25)) the median serum YKL-40was 157 μg/l and 12 of these patients (48%) had elevated serum YKL-40.Four patents had only visceral metastases and serum YKL-40 was abovenormal in 3 of these patients (75%).

FIG. 12 illustrates the individual serum YKL-40 concentrations inrelation to the presence of bone metastases on X-ray examination. Sinceserum YKL-40 levels are increased in patients with viscera metastases weonly evaluated the diagnostic value in patients without visceralinvolvement (N=41). Serum YKL-40 was significantly elevated (p<0.05) inpatients with .gtoreq.2 bone metastases compared to patients with onlyone or no bone metastasis. Four patients with a normal X-ray hadelevated serum YKL-40. However, two of these patients had a positivebone scanning and biopsies revealed bone marrow carcinosis, and othertwo developed radiographic bone metastases within 6 months.

Relating serum YKL-40 levels to the presence or absence of one or morebone metastases, YKL-40 levels were elevated in clinical group memberswith positive test results as opposed to negative test results. Inaddition, YKL-40 levels were elevated in positive test result memberswith more than one metastasis to bone as opposed to members with onemetastasis to bone (see, FIG. 10).

There was no clear relationship between the level of serum YKL-40 andother clinical parameters, such as the menopausal status of each patient(see Tables III and IV, below). However, serum YKL-40 values wereelevated compared to normal levels in 75% of the patients with visceralmetastasis and 48% of patients with metastases to bone. There also didnot appear to be any clear relationship between YKL-40 levels and age,although, as shown in FIG. 7, aberrant levels of YKL-40 did not appearin healthy (control group) women below age 70. Thus, particularly ascompared to other blood proteins measured (see Tables III-IV), YKL-40levels have diagnostic value with respect to metastases of breast cancercancer cells to bone and viscera.

6. Cox Regression Analysis

The initial Cox model included univariate significant blood tests andduration of recurrence free interval. Serum albumin was not includedbecause the value was only registered in 40% of the patients. Theinitial model showed that only serum YKL-40 and serum LDH wereindependent prognostic factors on survival after recurrence in the 60patients (Table III). Backward and forward elimination procedureseliminated all covariates except serum YKL-40 (p=0.001) and serum LDH(p=0.01). If only the 41 patients with first recurrence of breast cancerwere included in the calculations backward and forward eliminationprocedures again eliminated all covariates except serum YKL-40 (p=0.0004and serum LDH (p=0.037).

Based on the estimated survival pattern for the 4 combinations of thetwo serum YKL-40 levels and the two levels of serum LDH, the calculatedsurvival rate after 12 months for patients with normal serum LDH andnormal and elevated serum YKL-40 was 83% and 56%, respectively. Amongpatients with increase serum LDH levels the 12 months survival rate was67% for patients with normal and 28% for patients with high serumYKL-40.

TABLE III Serum YKL-40 in relation to different clinical parameters in60 women with first recurrence of breast cancer (cox univariate survivalanalysis). # of patients Median Survival P (log Variable Categories #alive Months (25%–75%) rank) Age ≦50 30 (8) 18 (10–55+) 0.04 Years >5030 (1) 16 (6–26) Menopausal pre- 30 (7) 18 (10–55+) 0.07 Status post- 29(2) 16 (6–26) Size of primary ≦2 25 (5) 22 (10–37) 0.46 tumor (cm) 3–416 (1) 16 (10–37) >4 18 (2) 12 (5–21) Axillary node negative 18 (5) 22(10–41+) 0.29 status positive 34 (3) 18 (10–41) Degree of low 13 (1) 12(4–18) 0.01 anaplasia high 15 (3) 26 (11–56) Estrogen receptor negative10 (2) 10 (6–18+) 0.99 status positive 18 (2) 21 (11–37) Recurrence Free≦24 32 (4) 13 (5–26) 0.18 interval (months) >24 28 (5) 21 (10–41)Dominant site of soft tissue 10 (4) 18 (6–50) 0.24 metastasis bone 25(1) 18 (12–26) viscera 19 (1)  9 (3–16) Blood Haemoglobin ≦7.0 11 (8)  9(2–16) 0.24 mmol/l >7.0 49 (1) 20 (10–41) Serum ASAT ≦30 38 (7) 20(10–47) 0.38 U/l >30 20 (2) 12 (4–26) Serum LDH ≦400 29 (8) 25 (15–53+)0.00 U/l >400 31 (1) 10 (5–21) Serum AP ≦275 40 (9) 22 (11–56) 0.00U/l >275 20 (0) 10 (3–18) Serum Albumin ≦600  8 (0)  7 (3–11) 0.00mg/l >600 16 (1) 23 (18–41) Serum ≦100 16 (2)  9 (2–16) 0.13 Prothrombin% >100 33 (4) 20 (10–35) Serum Ca⁺⁺ ≦1.35 13 (1) 12 (7–35) 0.24mmol/l >1.35  5 (0) 12 (2–18) Serum BGP ≦2 23 (5) 13 (4–56) 0.67 mmol/l2–2.9 19 (1) 18 (7–37) >2.9 18 (3) 24 (12–47) Serum YKL-40 ≦207 35 (9)24 (15–53+) 0.00 μg/ml >207 25 (0) 11 (6–21) All 60 (9) 16 (7–40)

TABLE IV Cox model for survival for patients entering staging ofrecurring breast cancer. Coefficient P (Wald's Covariate Categories βS.E. Test) Initial Model Serum YKL-40 ≦207, >207 1.04 0.36 0.00 (μg/l)Serum BGP ≦2, 2–2.9, >2.9 −0.20 0.19 0.31 (mmol/l) Serum ASAT ≦30, >30−0.25 0.33 0.44 (U/l) Serum LDII ≦400, >400 0.66 0.37 0.08 (U/l) SerumAP ≦275, >275 0.46 0.40 0.26 (U/l) Haemoglobin ≦7, >7 −0.03 0.42 0.94(mmol/l) Recurrence free ≦24, >24 0.25 0.31 0.41 interval (months) FinalModel* SerumYKL-40 ≦207, >207 1.11 0.33 0.00 (μg/l) Serum LDH ≦400, >4000.78 0.31 0.01 (U/l) *After backwared elimination (p value to remove:0.01; p value to enter: 0.15).

EXAMPLE IX Detection and Quantification of YKL-40 in Serum and Synoviaof Osteoarthritis Patients and Comparison to Levels Detected in Patientswith Knee Trauma

Accelerated metabolism of connective joint tissue is known to occur indegenerative joint disease (such as osteoarthritis), as well as ininflammatory joint diseases (such as RA). However, beyond thatsimilarity, the pathogenesis of degenerative and inflammatory jointdisease are dissimilar. For example, degenerative joint disease isbelieved to be associated with microfractures in bone caused by repeatedexposure to weight and impact loading. In healing, the fractures becomecovered with stiff and thickened subchondral bone that poorly absorbsthe energy associated with weight loading. The chondrocytes in articularcartilage are therefore subjected to pressure deflected from the bone,stimulating chondrocyte growth and metabolism. Initially, the body isable to make repairs as necessary, but eventually the degenerativeprocess predominates, resulting in loss of cartilage (see, e.g.,Harrison's Principles of Internal Medicine, 11th ed., McGraw-Hill, 1987,at page 1456. et seq.).

In contrast, the loss of cartilage in inflammatory joint disease isbelieved to be caused by inflammation of the synovia. Over time, thesynovia forms villi that will project into the joint cavity. Dispositionto RA in particular is believed to be conferred genetically, with an asyet unelucidated association with HLA-DR4, a class II majorhistocompatibility complex molecule. Inflammatory joint disease producesfairly specific clinical manifestations, including morning stiffness, asymptom that distinguishes inflammatory from non-inflammatory jointdisease (see, e.g., Harrison's Principles of Internal Medicine, supra atpage 1423, et seq.).

Given the differences in pathogenesis and pathology between inflammatoryand non-inflammatory joint disease, it would not be predictable that amarker for one would serve as a marker for the other. It was thereforesurprising to discover that YKL-40 levels are elevated to adiagnostically significant degree not only in body fluids of RApatients, but also in body fluids of osteoarthritis patients.

This discovery was made in a study comparing sera and synovia YKL-40levels from patients with traumatic knee injuries and osteoarthritis(late stage) to serum YKL-40 levels from healthy children and adults,the latter of which data confirmed baseline “normal” levels of serumYKL-40.

1. Control Group

476 normal children (aged 6-17 years; 236 girls and 240 boys)participated in the study. 275 adults (aged 18-79 years; 146 women and129 men) also participated in the study. Each participant was examinedand determined to be healthy according to conventional medicalstandards.

2. Patient Group

96 adults (aged 18-79 years; 37 women and 59 men) participated in thestudy. Clinically, these participants were grouped as follows: GroupA=12 patients who had knee pain leading to arthroscopy, but who weredetermined to be free of knee pathology; Group B=18 patients withoutsynovitis, but with an abnormal arthroscopy indicating trauma; GroupC=22 patients with mild to moderate synovitis; Group D=18 patients withacute severe synovitis; Group E=10 patients with chronic post-traumaticsynovitis; Group F=16 patients with osteoarthritis (late stage, knee).Serum was collected from each patient; synovia was only available insufficient quantity for analysis from 51 patients.

The patients were determined to be otherwise healthy according toconventional medical standards. None of the patients were takingmedication except 14 of the osteoarthritis patients, who were takingnon-steroidal anti-inflammatory drugs.

Serum and synovial fluid YKL-40 levels were determined as described inExample IV. Statistical analyses of assay results were performed using acommercially available software program to perform analyses according tostandard methods (SSPS software). Comparisons between groups werecalculated using the non-parametric Mann-Whitney and Kruskal-Wallistests known in the art. Correlations between the different parameterswere calculated using the Spearman rho test known in the art; p valuesof less than 0.05 were considered to be significant.

The data obtained from this study are set forth below.

TABLE V Serum YKL-40 concentrations (μg/l) in healthy subjects. AgeFemale Median Male Median Group (10–90% tile) (10–90% tile) Years N μg/lN μg/l 7–9 54 76 (60–113) 58 77 (57–105) 10–12 70 78 (61–107) 87 79(62–103) 13–15 78 82 (62–125) 70 83 (62–114) 16–17 34 90 (67–122) 25 86(72–122) All 236 79 (62–114) 240 80 (61–107) 18–19 9 75 7 94 20–29 20 95(71–122) 21 92 (75–137) 30–39 20 95 (54–141) 20 101 (75–154)  40–49 21100 (77–174)  17 124 (76–212)  50–59 29 111 (64–204)  25 125 (77–219) 60–69 21 101 (59–351)  24 111 (66–246)  70–79 24 168 (69–385)  — — All144 101 (69–205)  116 103 (75–213  

TABLE VI Characteristics of the patients. Clinical # Age Serum YKL-40 SFYKL-40 Diagnosis Serum Years (μg/l) (μg/l) Normal Knee^(§) 12/0  2786*** (72–292) — (18–55) Torn anterior 7/4 28 89** (66–117)  712*cruciate (26–34) (336–1358) ligaments Torn medial or 18/12 34 118**(72–233) 1407 lateral menisci (22–49) (312–9560) Early stage 17/10 50112** (29–315) 1306 knee osteo- (27–73) (500–7296) arthritis Late stageknee 16/15 71 195 (88–1648) 1944 osteo-arthritis (61–79) (279–5216)Chondro- 5/1 34 85** (60–187)  912 malacia (19–45) Osteo- 4/1 38 100*(76–158) 3540 chondrocyte (26–54) Acute severe 3/1 28 232 (107–287) 1928synovitis (23–31) Others^(§§) 14/7  23 83 (63–226)  878* (18–76)(201–2140) SF = Synovial fluid. Values are median (range). *p < 0.05 **p< 0.01 ***p < 0.001 vs. the level in patients with late stage kneeosteoarthritis. ^(§)Patients who had knee pain leading to arthroscopy,but in whom arthroscopy, radiologic studies of the knee, and bloodchemistry studies failed to reveal any pathologic condition related tothe knee pain. ^(§§)Patients with minor arthroscopic changes of the kneejoint. They had one of the following diagnoses: 1) Light or acuteposttraumatic synovitis (N = 8); 2) Slight defect of one of the menisci(N = 1); 3) Plicae synovitis (N = 3); 4) Mus articuli genus (N = 1); or5) Patella alignment (N = 1).

In summary, patients with late stage knee osteoarthritis hadsignificantly higher (1.5 to 2 times greater) levels of YKL-40 in seraand synovia as compared to other participants in the patient group andcontrol group (see, Tables V and VI). Approximately 10 times greaterlevels of YKL-40 were found in synovia as compared to sera, presumablyreflecting local production of YKL-40. Because cartilage is present inrelatively low amounts in the osteoarthritic knee, it may be assumedthat a significant proportion of the YKL-40 found in synovia issynthesized in the synovium, either through synovial cell hyperplasia orincreased secretion of YKL-40 by synovial cells. This hypothesis isfurther supported by the observation that YKL-40 levels weresignificantly higher (elevated 7 to 16 fold) in patients with severesynovitis as compared to patients with mild or no synovitis. However, itis also possible that YKL-40 is secreted into synovial fluid bychondrocytes. Whatever its source, it appears likely that, in contrastto cases of knee trauma, YKL-40 acts on matrix constituents to degradethem after onset of osteoarthritis or other connective tissue diseaseassociated with YKL-40.

EXAMPLE X Detection of Serum YKL-40 Levels in Sera from Patients withAlcoholic Cirrhosis and from Adults with Healthy Liver Tissue

39 adults participated in this study: 16 patients with normal liverfunction (7 women and 9 men; aged 35-74 years) and 23 patients withliver disease (8 women and 15 men; aged 31-75 years). Of the adults withnormal liver function, 7 were healthy, 7 had been diagnosed withhypertensive arterialis, 1 had been diagnosed with hypertensiverenovascularis, and 1 with ischaemia intestinalis. Of the adults withabnormal liver function, 18 had alcoholic cirrhosis with activehepatitis, 2 had alcoholic cirrhosis without active hepatitis, 2 hadhepatitis of unknown origin, and 1 had steatosis hepatitis. At the timeof the study, none of the patients were consuming alcohol.

After an overnight fast, the patients were catheterized under localanesthesia in the hepatic vein (from 20 patients with liver disease and14 with normal liver function) or the renal vein (17 patients with liverdisease and 13 patients with normal liver function). Blood samples wereobtained from the femoral artery and the organ vein in each patient thenanalyzed by radioimmunoassay as described in Example IV.

Using the data from the healthy adults reported in Example IX for acontrol reference, comparisons were made with the data collected fromthe above-described patients. The statistical analyses were performed asdescribed in Example IX. The arteriovenous extraction ratio wasdetermined as: E=C.sub.a-C.sub.v/C.sub.a, where C.sub.a is the YKL-40immunoreactivity in femoral arterial plasma and C.sub.v is the YKL-40immunoreactivity in plasma from the renal vein. The renal extractionratios of the cirrhotic patients and controls were not significantlydifferent; i.e., 0.051 (range=0.0076 to 0.14) and 0.028 (range=−0.0294to 0.3040), respectively. However, the patients with liver disease hadvery high YKL-40 levels compared to the levels detected in samples frompatients with normal liver function (p<0.001).

Median YKL-40 serum values detected in arterial and venous blood frompatients with liver disease are compared to median values detected inarterial and venous blood from patients with normal liver function. Asshown in Table VI, the former values were approximately 4 times greaterthan the latter values (p less than 0.001), strongly indicating thatYKL-40 is associated with degeneration of liver tissue.

TABLE VIII Plasma YKL-40 concentrations (μg/l) in patients with liverdisease compared to patients with normal liver function. Significance ofLiver Disease Controls difference Artery 495 106 p < 0.001 (101–2360) N= 20 (60–248) N = 14 Hepatic Vein 499*** 111** p < 0.001 (106–2568) N =20 (64–257) N = 14 Artery 493 125 p < 0.001 (100–2920) N = 17 (53–250) N= 13 Renal Vein 424*** 108* p < 0.001 (96–2760) N = 17 (48–243) N = 13*p < 0.05 **p < 0.01 ***p < 0.001

The invention being fully described, it will be apparent to those ofskill in the art that modifications may be made to the embodimentsdescribed above without departing from the spirit or scope of theinvention.

1. An isolated antibody that specifically binds YKL-40.
 2. The antibodyof claim 1, wherein said antibody is a monoclonal antibody.
 3. Theantibody of claim 1, wherein said antibody is a polyclonal antibody. 4.The antibody of any one of claims 1, 2, or 3, wherein said antibody islabeled with a detectable label.